枯草芽孢杆菌β-1,3-1,4-葡聚糖酶基因(bglS)在酵母菌中的克隆与表达

CLONING AND EXPRESSION OF A BACILLUS SUBTILIS β-1,3-1,4-GLUCANASE GENE(bgIS) IN SACCHROMYCES CEREVISIAE

将大肠杆菌质粒 pFG1中含枯草芽胞杆菌β-1,3-1,4-葡聚糖酶基因(bg1S)的2.7kb EcoRI 片段克隆到大肠杆菌/酵母菌穿梭质粒上组建成杂种质粒 YCSH,转化 S.cerevisiae并得到表达。两种不同方向的插入子(YCSH1和 YCSH 5)在酵母菌中表达出的β-1,3-1,4葡聚糖酶活性相差2.3倍。根据酶作用底物专一性测定和酶反应的最适 pH 分析表明:YCSH中 bg1S 基因产物与出发菌株 B.subtilis 1.88的基本酶学特性完全相同,但 YCSH 中 bg1S基因在酶母中的表达水平远比大肠杆菌中低。

A 2.7kb EcoRI DNA fragment carrying a Bacillus subtilis endo-β-1,3-1,4-glucanase gene(bg1S)from the E.coli plasmid pFG1.was cloned into an E.coli/S,cerevisiae shuttle vector toconstruct a hybrid plasmid YCSH.The hybrid plasmid was used to transform the Sacchromy-ces cerevisiae and expressed the β-1,3-1,4-glucanase activity.The difference of the β-1,3-1,4-glu-canase activities between two recombinants(YCSH1 and YCSH5)with two different insert orienorations was up to 2.3 fold.Analysis of substrate specificity and optimal pH of the enzyme show-ed that the β-glucanase enzyme coded by YCSH(bg1S)was identical with that found in Bacillussubtilis,but the expression level of the bg1S gene in S.cerevisiae(YCSH)was much lower thanthat in E.coli(YCSH).